O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

Improvement of learning and memory abilities and motor function in rats with cerebral infarction by intracerebral transplantation of neuron-like cells derived from bone marrow stromal cells

BACKGROUND: Transplantation of fetal cell suspension or blocks of fetal tissue can ameliorate the nerve function after the injury or disease in the central nervous system, and it has been used to treat neurodegenerative disorders induced by Parkinson disease. OBJECTIVE: To observe the effects of the transplantation of neuron-like cells derived from bone marrow stromal cells (rMSCs) into the brain in restoring the dysfunctions of muscle strength and balance as well as learning and memory in rat models of cerebral infarction. DESIGN: A randomized controlled experiment. SETTING: Department of Pathophysiology, Zhongshan Medical College of Sun Yat-sen University. MATERIALS: Twenty-four male SD rats (3-4 weeks of age, weighing 200-220 g) were used (Certification number:2001A027). METHODS: The experiments were carried out in Zhongshan Medical College of Sun Yat-sen University between December 2003 and December 2004. ① Twenty-four male SD rats randomized into three groups with 8 rats in each: experimental group, control group and sham-operated group. Rats in the experiment al group and control group were induced into models of middle cerebral artery occlusion. After in vitro cultured, purified and identified with digestion, the Fischer344 rMSCs were induced to differentiate by tanshinone ⅡA, which was locally injected into the striate cortex (18 area) of rats in the experimental group, and the rats in the control group were injected by L-DMEM basic culture media (without serum) of the same volume to the corresponding brain area. In the sham-operated group, only muscle and vessel of neck were separated. ② At 2 and 8 weeks after the transplantation, the rats were given the screen test, prehensile-traction test, balance beam test and Morris water-maze test. ③ The survival and distribution of the induced cells in corresponding brain area were observed with Nissl stained with toluidine blue and hematoxylin and eosin (HE) staining in the groups. MAIN OUTCOME MEASURES: ① Results of the behavioral tests (time of the Morris water-maze test screen test, prehensile-traction test, balance beam test); ② Survival and distribution of the induced cells. RESULTS: All the 24 rats were involved in the analysis of results. ① Two weeks after transplantation, rats with neuron-like cells grafts in the experimental group had significant improvement on their general muscle strength than those in the control group [screen test: (9.4±1.7), (4.7±1.0) s, P < 0.01]; forelimb muscle strength [prehensile-traction test: (7.6±1.4), (5.2±1.2) s, P < 0.01], ability to keep balance [balance beam test: (7.9±0.74), (6.1±0.91) s, P < 0.01] and abilities of learning and memory [latency to find the platform: (35.8±5.9), (117.5±11.6) s, P < 0.01; distance: (623.1±43.4), (1 902.3±98.6) cm, P < 0.01] as compared with those in the control group. The functional performances in the experimental group at 8 weeks were better than those at two weeks, which were still obviously different from those in the sham-operated group (P < 0.05). ② The HE and Nissl stained brain tissue section showed that there was nerve cell proliferation at the infarcted cortex in the experiment group, the density was higher than that in the control group, plenty of aggregative or scattered cells could be observed at the site where needle was inserted for transplantation, the cells migrated directively towards the area around them, the cerebral vascular walls were wrapped by plenty of cells; In the control group, most of the cortices were destroyed, karyopyknosis and necrosis of neurons were observed, normal nervous tissue structure disappeared induced by edema, only some nerve fibers and glial cells remained. CONCLUSION: The rMSCs transplantation can obviously enhance the motor function and the abilities of learning and memory in rat models of cerebral infarction.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

Objective To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardiomyocytes. Methods Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract (IMTE), normal myocardial tissue extract (NMTE) and cultured neonatal myocardial lysate (NML)] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction (RT-PCR) detection, immunocytochemical analysis, and transmission electron microscopy. Results After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Conclusions Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

Gene expression profiles associated with osteoblasts differentiated from bone marrow stromal cells

Objective:To study the changes of gene expression profiles associated with osteoblasts differentiated from rat bone marrow stromal cells in vitro by gene chip technique.Methods:rat Rone marrow stromal cells were isolated and cultured,and differentiation was induced by dexamethasone,β-glycerol phosphate and vitamin C.Cellular mRNA was extracted and reverse transcribed into cDNA,thus related genes expression differences were detected by gene expression profile chip.Results:Calcifying nodules were visible in the induced cells.There were27.7%genes expressed differentially,three times more than the normal and induced cells,and some genes were related to transcription,translation,glycosylation modification.Extracellular matrix,signal molecules and metabolism were up—regulated.Conclusions:The gene chip technique can be used to detect the multi-gene different expression in the differentiationinduceed rat BMSCs,and these differentially expressed genes are necessary genes related to rat BMSCs proliferation and induction of osteoblastic differentiation.

Improvement of neurological function in rats with spinal cord injury after the transplantation of neural stem cells directly differentiated from bone marrow mesenchymal stem cells

Objective To study the effect and mechanism of neurological function recovery in rats with spinal cord injury ( SCI) rats after transplantation of neural stem cells which are directly differentiated from bone marrow mesenchymal stem cells ( BMSC ) ,and to investigate the suitable engraftment time. Methods BMSC at 3rd passage were differentiated into neural stem cells ( NSC) , and immunofluorescence staining was used to

Intra-portal transplantation of bone marrow stromal cells ameliorates liver fibrosis in mice

BACKGROUND:Bone marrow cells can differentiate into hepatocytes in a suitable microenvironment.This study was undertaken to investigate the effects of transplanted bone marrow stromal cells (BMSCs) on liver fibrosis in mice. METHODS:BMSCs were harvested and cultured from male BALB/c mice, then transplanted into female syngenic BALB/c mice via the portal vein. After partial hepatectomy, diethylnitrosamine (DEN) was administered to induce liver fibrosis. Controls received BMSCs and non-supplemented drinking water, the model group received DEN with their water, and the experimental group received BMSCs and DEN. Mice were killed after 3 months, and ALT, AST, hyaluronic acid (HA), and laminin (LN) in serum and hydroxyproline (Hyp) in the liver were assessed. Alpha-smooth muscle actin (α-SMA) in the liver was assessed by immunohistochemistry. Bone marrow- derived hepatocytes were identified by fluorescent in situ hybridization (FISH) in liver sections. RESULTS:BMSCs were shown to differentiate into hepatocyte-like phenotypes after hepatocyte growth factor treatment in vitro. Serum ALT, AST, HA, and LN were markedly reduced by transplanted BMSCs. Liver Hyp content and α-SMA staining in mice receiving BMSCs were lower than in the model group, consistent with altered liver pathology. FISH analysis revealed the presence of donor- derived hepatocytes in the injured liver after cross-gender mouse BMSC transplantation. After three months, about 10% of cells in the injured liver were bone marrow-derived. CONCLUSION:BMSCs transplanted via the portal vein can convert into hepatocytes to repair liver injury induced by DEN, restore liver function, and reduce liver fibrosis.

Bone Marrow Stromal Cells Express Neural Phenotypes in vitro and Migrate in Brain After Transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC (hBMSC) to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilament1 (NF1), nestin and neuron-specific enolase (NSE) in rat BMSC (rBMSC). Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NF1 and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated. rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.

Synergistic effects of brain-derived neurotrophic factor and retinoic acid on inducing the differentiation of bone marrow stromal cells into neuron-like cells in adult rats in vitro

BACKGROUND: Under induction of retinoic acid (RA), bone marrow stromal cells (BMSCs) can differentiate into nerve cells or neuron-like cells, which do not survive for a long time, so those are restricted to an application. Other neurotrophic factors can also differentiate into neuronal cells through inducing BMSCs; especially, brain-derived neurotrophic factor (BDNF) can delay natural death of neurons and play a key role in survival and growth of neurons. The combination of them is beneficial for differentiation of BMSCs. OBJECTIVE: To investigate the effects of BDNF combining with RA on inducing differentiation of BMSCs to nerve cells of adult rats and compare the results between common medium group and single BDNF group. DESIGN: Randomized controlled animal study.SETTING: Department of Neurology, Affiliated Hospital of Xuzhou Medical College. MATERIALS: The experiment was carried out in the Clinical Neurological Laboratory of Xuzhou Medical College from September 2003 to April 2005. A total of 24 SD rats, of either gender, 2 months old, weighing 130-150 g, were provided by Experimental Animal Center of Xuzhou Medical College [certification: SYXK (su) 2002-0038]. Materials and reagents: low-glucose DMEM medium, bovine serum, BDNF, RA, trypsin, separating medium of lymphocyte, monoclonal antibody of mouse-anti-nestin, neuro-specific enolase, glial fibrillary acidic protein (GFAP) antibody, SABC kit, and diaminobenzidine (DAB) color agent. All these mentioned above were mainly provided by SIGMA Company, GIBCO Company and Boshide Company. METHODS: Bone marrow of SD rats was selected for density gradient centrifugation. BMSCs were undertaken primary culture and subculture; and then, those cells were induced respectively in various mediums in total of 3 groups, including control group (primary culture), BDNF group (20 μg/L BDNF) and BDNF+RA group (20 μg/L BDNF plus 20 μg/L RA). On the 3rd and the 7th days after induction, BMSCs were stained immunocytochemically with nestin (sign of nerve stem cells), neuron-specific enolase (NSE, sign of diagnosing neurons) and GFAP (diagnosing astrocyte), and evaluated cellular property. MAIN OUTCOME MEASURES: Induction and differentiation in vitro of BMSCs in 3 groups. RESULTS: ① Induction and differentiation of BMSCs: Seven days after induction, cells having 2 or more apophyses were observed. Soma shaped like angle or erose form, which were similar to neurons and glial cells having strong refraction. ② Results of immunocytochemical detection: Three days after induction, rate of positive cells in BDNF+RA group was higher than that in BDNF group and control group [(86.15±4.58)%, (65.43±4.23)%, (4.18±1.09)%, P < 0.01]. Seven days after induction, rate of positive cells was lower in BDNF group and BDNF+RA group than that in both groups at 3 days after induction [(31.12±3.18)%, (29.35±2.69)%, P < 0.01]; however, amounts of positive cells of NSE and GFAP were higher than those at 3 days after induction (P < 0.01); meanwhile, the amount in BDNF+RA group was remarkably higher than that in BDNF group (P < 0.01). CONCLUSION: Combination of BDNF and RA can cooperate differentiation of BMSCs into neurons and astrocyte, and the effect is superior to single usage of BDNF.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison, the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

Adipose-derived stromal cells resemble bone marrow stromal cells in hepatocyte differentiation potential in vitro and in vivo

AIM To investigate whether mesenchymal stem cells(MSCs) from adipose-derived stromal cells(ADSCs) and bone marrow stromal cells(BMSCs) have similar hepatic differentiation potential.METHODS Mouse ADSCs and BMSCs were isolated and cultured. Their morphological and phenotypic characteristics, as well as their multiple differentiation capacity were compared. A new culture system was established to induce ADSCs and BMSCs into functional hepatocytes. Reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analyses were performed to identify the induced hepatocytelike cells. CM-Dil-labeled ADSCs and BMSCs were then transplanted into a mouse model of CCl4-induced acute liver failure. fluorescence microscopy was used to track the transplanted MSCs. Liver function was tested by an automatic biochemistry analyzer, and liver tissue histology was observed by hematoxylin and eosin(HE) staining.RESULTS ADSCs and BMSCs shared a similar morphology and multiple differentiation capacity, as well as a similar phenotype(with expression of CD29 and CD90 and no expression of CD11 b or CD45). Morphologically, ADSCs and BMSCs became round and epithelioid following hepatic induction. These two cell types differentiated into hepatocyte-like cells with similar expression of albumin, cytokeratin 18, cytokeratin 19, alpha fetoprotein, and cytochrome P450. fluorescence microscopy revealed that both ADSCs and BMSCs were observed in the mouse liver at different time points. Compared to the control group, both the function of the injured livers and HE staining showed significant improvement in the ADSC-and BMSC-transplanted mice. There was no significant difference between the two MSC groups.CONCLUSION ADSCs share a similar hepatic differentiation capacity and therapeutic effect with BMSCs in an acute liver failure model. ADSCs may represent an ideal seed cell type for cell transplantation or a bio-artificial liver support system.

Study on the adoption of Schwann Cell Phenotype by Bone Marrow Stromal Cells in vitro and in vivo

Objective To explore the possibilities of bone marrow stromal cells (MSCs) to adopt Schwann cell phenotype in vitro and in vivo in SD rats. Methods MSCs were obtained from tibia and femur bone marrow and cultured in culture flasks. Beta-mercaptoethanol followed by retinoic acid, forskolin, basic-FGF, PDGF and heregulin were added to induce differentiation of MSCs’. Schwann cell markers, p75, S-100 and GFAP were used to discriminate induced properties of MSCs’ by immunofluorescent staining. PKH-67-labelled MSCs were transplanted into the mechanically injured rat sciatic nerve, and laser confocal microscopy was performed to localize the PKH67 labelled MSCs in the injured sciatic nerve two weeks after the operation. Fluorescence PKH67 attenuation rule was evaluated by flow cytometry in vitro. Results MSCs changed morphologically into cells resembling primary cultured Schwann cells after their induction in vitro. In vivo, a large number of MSCs were cumulated within the layer of epineurium around the injured nerve and expressed Schwann cell markers, p75, S-100, and GFAP. Conclusion MSCs are able to support nerve fiber regeneration and re-myelination by taking on Schwann cell function, and can be potentially used as possible substitutable cells for artificial nerve conduits to promote nerve regeneration.

Bone marrow stromal cell versus neural stem cell transplantation in a C6 glioma rat model

BACKGROUND: Embryonic neural stem cells (NSCs) have provided positive effects for the treatment of glioma. However, the source for embryonic NSCs remains limited and high amplification conditions are required. Bone marrow stromal cells (BMSCs) have been proposed for the treatment of glioma. OBJECTIVE: To investigate biological changes in NSCs and BMSCs following transplantation into rat models of glioma. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the Embryonic Stem Cell Research Laboratory of Yunyang Medical College from February 2006 to August 2008. MATERIALS: The rat C6 glioma cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; mouse anti-bromodeoxyuridine (BrdU) monoclonal antibody and Cy3-labeled goat anti-mouse IgG antibody was purchased from Upstate, USA. METHODS: A total of 95 Sprague Dawley rats were randomly assigned to three groups: NSC (n = 35), transplanted with > 6 × 106 NSCs via left medial hind limb; BMSC (n = 35), transplanted with > 1 × 106 BMSCs via left medial hind limb; model group (n = 25), injected with the same volume of 0.1 mmol/L phosphate buffered saline. MAIN OUTCOME MEASURES: Gliomal growth and size were assessed by nuclear magnetic resonance, and glioma morphological features were observed following hematoxylin-eosin staining and BrdU immunohistochemistry 3 and 4 weeks following transplantation. RESULTS: The average survival of rats in the BMSC, NSC, and model groups was 4.03, 4.28, and 3.88 weeks. At 3 weeks, there was no significant difference in the average glioma diameter between the BMSC and model groups (P > 0.05). However, gliomal diameter was significantly decreased in the NSC group compared with the model group (P < 0.05). At 4 weeks, there was no statistical difference between the groups (P > 0.05). BrdU immunohistochemistry revealed that BMSCs and NSCs appeared to migrate to the gliomas. CONCLUSION: NSCs inhibited glioma cell growth and prolonged rat survival. BMSCs did not significantly suppress glioma cell growth.

Neuronal differentiation effects of vascular endothelial factor on bone marrow stromal cells

BACKGROUND:Studies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However,very few inducers of BMSC differentiation have been used in clinical application.The effects of vascular endothelial growth factor (VEGF) on in vitro neuronal differentiation of BMSCs remain poorly understood.OBJECTIVE:To investigate the effect of VEGF on neuronal differentiation of BMSCs in vitro,and to determine the best VEGF concentration for experimental induction.DESIGN,TIME AND SETTING:In vitro comparative study was performed at the Central Laboratory and Laboratory of Male Reproductive Medicine,Shenzhen Hospital of Peking University from October 2008 to August 2009.MATERIALS:Recombinant human VEGF165 was purchased from Peprotech Asia,Rehovot,Israel.Neuron-specific enolase (NSE) was purchased from Beijing Biosynthesis Biotechnology,China.METHODS:BMSCs were harvested from adult Sprague Dawley rats.The passaged cells were pre-induced with 10 ng/mL basic fibroblast growth factor for 24 hours,followed by differentiation induction with 0,5,10,and 20 ng/mL VEGF,respectively.MAIN OUTCOME MEASURES:Morphological changes in BMSCs prior to and following VEGF induction.Expression of NSE following induction was determined by immunocytochemistry.RESULTS:Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20 ng/mL VEGF.However,these changes were not observed in the control group.At 10 days after induction,the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group (P < 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction (P < 0.05).Moreover,the number of NSE-positive cells was greater at 10 days compared with at 3 days after induction (P < 0.05).CONCLUSION:Of the VEGF concentrations tested,10 ng/mL induced the greatest number of neuronal-like cells in vitro from BMSCs.

Mechanism of in Vitro Differentiation of Bone Marrow Stromal Cells into Neuron-like Cells

Summary: In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced into neural stem cells, and then differentiated into neuron-like cells in vitro.

Intravenous transplantation of bone marrow mesenchymal stem cells promotes neural regeneration after traumatic brain injury

To investigate the supplement of lost nerve cells in rats with traumatic brain injury by intravenous administration of allogenic bone marrow mesenchymal stem cells, this study established a Wistar rat model of traumatic brain injury by weight drop impact acceleration method and administered 3 × 106 rat bone marrow mesenchymal stem cells via the lateral tail vein. At 14 days after cell transplantation, bone marrow mesenchymal stem cells differentiated into neurons and astrocytes in injured rat cerebral cortex and rat neurological function was improved significantly. These findings suggest that intravenously administered bone marrow mesenchymal stem cells can promote nerve cell regeneration in injured cerebral cortex, which supplement the lost nerve cells.

Experimental study on the induction of bone marrow stromal cells differentiating into cardiomyocyte-like cells with cardiomyocytes in vitro

Objective: To investigate the feasibility of bone marrow stromal cells (BMSCs) differentiating into cardiomyocyte-like cells in heterogeneous cardiomyocytes microenvironment in vitro. Methods: Mouse GFP-BMSCs were isolated by centrifugation through a Ficoll step gradient and purified by plating culture and depletion of the non-adherent cells. Neonatal rat cardiomyocytes (CMs) were isolated by enzymatic dissociation from hearts of 1-to 2-day-old Sprague-Dawley (SD) rats and differentially plated to remove fibroblasts. Mouse GFP-BMSCs were cocultured with neonatal rat CMs through direct and indirect contact, respectively. Cardiomyogenic differentiation of BMSCs was evaluated by immunostaining with anti -α-actin monoclonal antibody and observing synchronous contraction with adjacent CMs by phase contrast microphotography. Results: On day 7 of coculture, GFP-BMSCs (CMs : BMSCs = 4 : 1)attached to nonfluorescent contracting cells (rat-derived CMs) showed myotube-like formation and started to contract synchronously with adjacent cardiomyocytes. About 10% of the fluorescent GFP-BMSCs were cardiomy-ocyte-like cells as determined by cell morphology and positive actin staining. Conclusion:Direct cell-to-cell interaction with CMs is crucial for cardiomyogenic differentiation of BMSCs in heterogeneous CMs microenvironment in vitro. This provides a novel inducing pathway for directional differentiation of cardiovascular tissue engineering seed cells.

Promotion of Chondrogenesis of Marrow Stromal Stem Cells by TGF-β3 Fusion Protein In Vitro

The purpose of this study was to investigate the repair of the osteoarthritis(OA)-induced cartilage injury by transfecting the new TGF-β3 fusion protein(LAP-MMP-mTGF-β3) with targeted therapy function into the bone marrow-derived mesenchymal stem cells(MSCs) in rats. The recombinant of pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, so as to construct the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3. pIRESEGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cultured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models.The OA animal models were surgically induced by anterior cruciate ligament transaction(ACLT). The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Green and graded by Mankin's scale. pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein(39 kD) was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.

Bone morphogenetic protein-7 induced bone marrow stromal cells differentiate into neuron-like cells

Bone morphogenetic protein-7 is widely accepted as an inducer for bone marrow stem cells differ-entiating into osteoblasts and chondrocytes. Whether bone marrow stromal cells differentiate into neuron-like cells remains unclear. The current study examined the presence of positive cells for in-termediate filament protein and microtubule associated protein-2 in the cytoplasm of bone marrow stromal cells induced by bone morphogenetic protein-7 under an inverted microscope, while no expression of glial fibrillary acidic protein was found. Reverse transcription PCR electrophoresis also revealed a positive target band for intermediate filament protein and microtubule-associated protein 2 mRNA. These results confirmed that bone morphogenetic protein-7 induces rat bone marrow stromal cells differentiating into neuron-like cells.